Hepatic fibrogenesis almost exclusively occurs in a proinflammatory environment. Proinflammatory signaling pathways are believed to contribute to the activation of HSCs, the major cellular source of extracellular matrix in the fibrotic liver. While TGF and PDGF drive the activation and proliferation of HSCs, their actions cannot account for the inflammatory component of hepatic fibrogenesis. It remains unclear (i) which mediators trigger inflammatory signals in hepatic fibrogenesis and (ii) how fibrogenic and inflammatory signaling pathways cooperate in HSC activation. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and are among the most potent inducers of inflammation. Patients with hepatic fibrosis display increased levels of the TLR4 ligand lipopolysaccharide (LPS) due to decreased intestinal motility, alterations of the intestinal bacterial flora and increased bacterial translocation. Moreover, potential endogenous TLR ligands such HMGB1 and hyaluronan, are upregulated in the injured liver. Kupffer cells (KCs) and HSCs are both highly responsive to TLR ligands rendering them the most likely candidates to mediate fibrogenesis in response to the ligands. We hypothesize that TLRs enable HSCs and KCs to sense molecular patterns associated with liver injury and trigger signals that are required for HSC activation and fibrogenesis. Using bile duct ligation and CCl4 treatment as models of experimental fibrogenesis, we will define the contribution of TLR2, TLR4 and TLR9 to hepatic fibrosis in the respective knockout mice and test whether TLR antagonists reduce hepatic fibrogenesis. Moreover, we will determine the cellular targets that promote TLR-dependent fibrogenesis in TLR-chimeric mice and in mice challenged with TLR ligands (Aim 1). We will define the contribution of bacterial TLR ligands to hepatic fibrogenesis using germ-free mice, mice mono-associated with wild-type and LPS-deficient bacteria, and mice with defects in LPS signaling. The contribution of endogenous TLR ligands will be investigated by blocking HMGB1 and hyaluronan during experimental fibrogenesis (Aim 2). Finally we will determine mechanisms by which TLR ligands promote HSC activation and fibrogenesis with a specific focus on TGF and PDGF pathways (Aim 3). The pursuit of these three aims will allow us to determine how TLRs contribute to hepatic fibrogenesis and to possibly develop clinically feasible strategies for the prevention or treatment of hepatic fibrosis. Public Health Relevance: Hepatic fibrosis affects several million people in the United States and progresses to hepatic cirrhosis and hepatocellular cancer in many patients. The goal of this proposal is to investigate the role of Toll-like receptors and their ligands in this process, and to evaluate whether targeting Toll-like receptors or sources of Toll-like receptor ligands such as the intestinal bacterial flora may be a useful strategy for the prevention or treatment of hepatic fibrosis.